Skip to Content
Merck
  • Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

PloS one (2015-09-04)
Stefanie Jeruschke, Kay Jeruschke, Andrew DiStasio, Sinem Karaterzi, Anja K Büscher, Perihan Nalbant, Ludger Klein-Hitpass, Peter F Hoyer, Jürgen Weiss, Rolf W Stottmann, Stefanie Weber
ABSTRACT

Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far. To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2bbrdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm's tumour 1, Podocin, Nephrin and Synaptopodin. Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methanol, suitable for HPLC
Sigma-Aldrich
Methanol, JIS special grade, ≥99.8%
Sigma-Aldrich
Hydrogen peroxide solution, SAJ first grade, ≥30.0%
Sigma-Aldrich
Anti-THBS1 antibody produced in mouse, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Anti-DCDC2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Glutaraldehyde solution, SAJ first grade, 20.0-26.0%
Sigma-Aldrich
Methanol, SAJ first grade, ≥99.5%
Sigma-Aldrich
Methanol, JIS 300, ≥99.8%, for residue analysis
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 50% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 8% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Glutaraldehyde solution, Grade I, 70% in H2O, specially purified for use as an electron microscopy fixative or other sophisticated use
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Hydrogen peroxide solution, 30 % (w/w) in H2O, contains stabilizer
Sigma-Aldrich
DL-Glyceraldehyde 3-phosphate solution, 45-55 mg/mL in H2O
Sigma-Aldrich
Glutaraldehyde solution, Grade II, 25% in H2O
Sigma-Aldrich
Glutaraldehyde solution, 50% in H2O, suitable for photographic applications
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
Vinylene carbonate, contains ≤2% BHT as stabilizer, 97%
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Glutaric dialdehyde solution, 50 wt. % in H2O, FCC
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, 99.93%
Sigma-Aldrich
Hydrogen peroxide solution, contains potassium stannate as inhibitor, 30-32 wt. % in water, semiconductor grade, 99.999% trace metals basis
Sigma-Aldrich
Glutaraldehyde solution, 50 wt. % in H2O
Sigma-Aldrich
Hydrogen peroxide solution, 34.5-36.5%
Sigma-Aldrich
Methanol, SAJ special grade
Sigma-Aldrich
Anti-GAPDH antibody, Mouse monoclonal, clone GAPDH-71.1, purified from hybridoma cell culture
Sigma-Aldrich
Methanol-12C, 99.95 atom % 12C